This research program seeks to acquire new information about the regulation of the delivery of retinol to vitamin A-requiring tissues. Progress in this research area has been seriously limited by a total lack of information about the molecular events involved in the formation of the vitamin A-retinol-binding protein (RBP) complex within the liver and about the subcellular anatomic locus of these events. A multiple approach has been proposed to study these problems. The initial project is designed to define the subcellular site where retinol complexes with RBP in the liver cell. Radioactive retinyl palmitate, with a very high specific activity, will be administered to vitamin A deficient rats, and Triton X-100 extracts of the subfractions of microsomes and Golgi apparatus will be examined by gel filtration chromatography to determine where the retinol-RBP complex is formed. The aim of a second project is to develop an assay with the capability to distinguish between intact and partially degraded RBP, so that the actual levels of intact RBP can be accurately measured. This assay will involve the immunoprecipitation of RBP and separation of the intact and partially degraded RBP by SDS polyacrylamide electrophoresis. This assay will aid in the determination of the subcellular location of the block in the secretion of RBP in vitamin A deficient animals. In the third project retinyl acetate, an enzyme that has a very high probability of regulating the rate of the formation of the retinol-RBP complex, will be isolated and will be characterized with regard to its physical and kinetic properties. In addition studies will be undertaken to determine the subcellular location of retinyl acetate hydrolase within the liver cell and to relate the activity of this enzyme to the rate of mobilization of vitamin A from the liver. Taken together the informaton developed in these studies should define, in part, the mechanisms which control the mobilization of vitamin A from its liver stores, as well as some of the factors which determine and regulate the plasma concentration of vitamin A. These studies should also provide useful new insights into the mechanisms involved in the secretion of plasma proteins in general.